Positron Emission Tomography (PET), a molecular imaging technology, detects a myriad of diseases non-invasively. PET imaging systems create images based on the distribution of positron-emitting isotopes in the tissue of the patient. The isotopes are typically administered to a patient by injection of probe molecules that are comprised of a positron-emitting isotope, such as F-18, C-11, N-13 or O-15, covalently attached to a molecule that metabolizes or localizes in the body, or that binds to receptor sites within the body.
One of the most widely used positron-emitter labeled PET molecular imaging probes is 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). [18F]FDG, which primarily targets glucose transporters, is an accurate clinical tool for the early detection, staging, and restaging of cancer. PET-FDG imaging is increasingly used to monitor cancer chemo- and radio-therapy because early changes in glucose utilization have been shown to correlate with outcome predictions. A characteristic feature of tumor cells is their accelerated glycolysis rate, which results from the high metabolic demands of rapidly proliferating tumor tissue. Like glucose, FDG is taken up by cancer cells via glucose transporters and is phosphorylated by hexokinase to FDG-6 phosphate. The latter cannot proceed any further in the glycolysis chain or leave the cell, due to its charge, allowing cells with high glycolysis rates to be detected.
Although useful in many contexts, several limitations of FDG-PET imaging for monitoring cancer exist as well. Accumulation of FDG in inflammatory tissue limits the specificity of FDG-PET. Conversely, nonspecific FDG uptake may also limit the sensitivity of PET for tumor response prediction. Therapy-induced cellular stress reactions have been shown to cause a temporary increase in FDG-uptake in tumor cell lines treated by radiotherapy and chemotherapeutic drugs. Furthermore, physiologically-high normal background activity (e.g. FDG uptake in the brain) can render the quantification of cancer-related FDG-uptake impossible in some areas of the body.
Due to these limitations, other PET imaging tracers are being developed to target a variety of enzyme-mediated transformations in cancer tissue, such as 6-[F-18]-fluoro-L-DOPA for dopamine synthesis, 3′-[F-18]Fluoro-3′-deoxythymidine (FLT) for DNA replication, and [C-11](methyl)choline for choline kinase, as well as ultra-high specific activity receptor-ligand binding (e.g., 16α [F-18]fluoroestradiol). Molecularly targeted agents have demonstrated great potential value for the non-invasive PET imaging of specific metabolic targets in cancers. Despite the clear clinical value of incorporating PET imaging into patient management, limitations do exist. In certain instances, current imaging probes lack specificity or have inadequate signal to background characteristics. In addition, new biological targets that are being tested for therapeutic intervention will require new imaging probes to evaluate their therapeutic potential. Additional biomarkers are needed that show a very high affinity to, and specificity for, tumor targets to support cancer drug development and to provide health care providers with a means to accurately diagnose disease and monitor treatment.
The carbonic anhydrases (CAs, EC 4.2.1.1) form a large family of genes encoding zinc metalloenzymes of great physiological importance. As catalysts for the reversible hydration of carbon dioxide, these enzymes participate in many diverse biological processes, including respiration, calcification, acid-base balance, bone resorption and in the formation of both cerebrospinal fluid and gastric acid. As a reflection of the importance of these enzymes, the carbonic anhydrases are widely distributed in many different living organisms. In higher vertebrates, including humans, 16 isozymes have been identified so far that differ in their subcellular localization, catalytic activity and susceptibility to different classes of inhibitors. Some of these isozymes are cytosolic (CA-I, CA-II, CA-III, CA-VII and CA-XIII), others are membrane bound (CA-IV, CA-IX, CA-XII and CA-XIV), two are mitochondrial (CA-VA and CA-VB), and one is secreted in saliva (CA-VI). The CAs and CA-related proteins show extensive diversity in their tissue distribution, levels of expression, and putative or established biological functions. Some CAs are ubiquitously expressed in almost all tissues, such as CA-II, while the expression of other CAs appear to be restricted in their tissue expression patterns.
Recently, it has been shown that two CA isozymes (CA-IX and CA-XII) are prominently associated with, and over expressed in, many tumors, where they are involved in crucial processes connected with cancer progression. The first CA found to be associated with cancers was CA-IX, as reported in 1992 (Pastorekova S., et al., Virology, 1992, 187, 620-626). The strong association between CA-IX expression and intratumorial hypoxia has been demonstrated in the cervical, breast, head and neck, bladder and non-small cell lung carcinomas. In addition, in breast carcinomas and non-small cell lung carcinomas, correlation between CA-IX and a constellation of proteins involved in angiogenesis, apoptosis inhibition and cell-cell adhesion disruption has been observed. Hypoxia is linked with acidification of the extracellular milieu that facilitates tumor invasion and CA-IX is believed to play a role in this process via its catalytic activity. Thus, there are several reasons that CA-IX is considered as one of the best targets for cancer diagnosis and therapy. For instance, CA-IX is an integral plasma membrane protein with an extracellularly exposed enzyme active site. Also, CA-IX has a very high catalytic activity with the highest proton transfer rate among the known CAs. In addition, CA-IX is present in few normal tissues such as the gall bladder and stomach, but its over expression is strongly associated with many tumor tissues such as lung, head and neck, renal and cervical carcinomas. Finally, CA-IX levels dramatically increase in response to hypoxia via a direct transcriptional activation of the CA-IX gene by HIF-1 (Giatromanolaki et al., Cancer Res., 2001, 61, 7992-7998; Dubois et al., Br. J. Cancer, 2004, 91, 1947-1954), and the expression of CA-IX in certain tumors can be a sign of poor prognosis. Consequently, discovery of specific inhibitors for CA-IX constitutes a novel approach to the diagnosis and treatment of cancers in which CA-IX is expressed.
The enzymatic activity of carbonic anhydrases can be efficiently blocked by sulfonamide inhibitors, a fact that has been therapeutically exploited in diseases caused by excessive activities of certain CA isoforms (e.g. CA-II in glaucoma). There is also experimental evidence that sulfonamides block both tumor cell proliferation and invasion in vitro, and tumor growth in vivo, but the targets of those sulfonamides have not yet been identified. Unfortunately, the sulfonamides available thus far indiscriminately inhibit various CA isoenzymes and the sulfonamides' lack of selectivity compromises their clinical utilization presenting a major drawback for the application of sulfonamides in specific CA-IX-targeted therapies.
Currently, there exists very few reported PET imaging agents that are both selective for the CA-IX enzyme and which have provided useful in vivo images. For example, the use of the radiolabeled monoclonal antibody 124I-G250 for targeting CA-IX in hypoxic tumors and CA-IX expression in xenografted human renal cell carcinoma animal models (Lawrentschuk, N., et al, British Journal of Urology, 2006, 97, Suppl. 1, 10-10(1)) has been reported. However, it is well documented that the application of monoclonal antibodies has substantial limitations, such as slow clearance.